1. Field of the Invention
This invention relates to methods for controlling the proliferation of smooth muscle cells including vascular smooth muscle cells. Proliferation can be inhibited by treatment of smooth muscle cells with agents that inactivate a novel transcription factor, a representative member of a new class of rel-related factors. Stimulating the activity of this same factor can induce smooth muscle cells to multiply and proliferate. The invention also relates to purified smooth muscle cell transcription factor, to compositions containing purified transcription factor or transcription factor inhibitors and inducers, and to methods for the treatment of diseases and disorders associated with cell proliferation and the activity of members of this new class of transcription factors.
2. Description of the Background
Arteries are divided into three distinct categories based on their overall size. Large arteries comprise the elastic arteries such as the aorta, the innominate, the subclavian, the beginning of the common carotid and the origins of the pulmonary arteries. Medium sized arteries comprise the muscular arteries and, unlike the larger arteries, have fairly well-defined, layered walls. The smaller arteries include the arterioles and those vessels with an inner diameter of less than 0.2 mm. Regardless of size, each type of artery has a similar structure comprising an outer tunica adventitia, a media tunica adventitia, and an inner tunica intima. The tunica adventitia is relatively thick in the larger sized arteries and contains connective tissue in which elastic fibers, nutrient vessels (vasa vasorum) and nerves can be found. The tunica media is the muscular layer. It is rich in elastic tissues, especially in the larger arteries, and predominantly composed of circular or spiral smooth muscle cells (SMCs) arranged in concentric layers. The outer limit of this layer is marked with an external elastic membrane which is less well developed than the corresponding internal membrane.
The medium-sized arteries are principally the muscular arteries and the vessels sometimes referred to as the distributor arteries. These vessels permeate the muscles and organs of the body and have fairly well-defined cellular layers. The tunica adventitia of medium sized arteries is a defined layer of connective tissue in which elastic and nerve fibers are dispersed reflecting the role these vessels play in the autonomic regulation of blood flow. Small arteries have a progressive loss of external elastic membrane, such that the definitions between the layers of cells are lost. As vessel size approaches the arterioles, vessel walls comprise an endothelial lining of subendothelial connective tissue, a layer of muscular media and a small collagenous adventitia. Despite their small size, arterioles are richly supplied with nervous connections to the autonomic system and constitute the majority site of autonomic control of vascular blood flow. In this capacity, the smaller arteries and arterioles bear the brunt of elevations of blood pressure by alterations in their structure.
The main components of the vascular wall play important roles in all types of vascular pathologies. The single layer of continuous endothelium lining arteries and veins forms a unique thrombo-resistant layer between blood and potentially thrombogenic subendothelial tissues. The integrity of this layer is fundamental for maintaining normal structure and function of the entire vessel wall. Endothelial injury may be, in part, responsible for the initiation of atherosclerosis and the vascular lesions produced by hypertension.
Vascular SMCs have recently been demonstrated to possess a great many functions. These cells, rather than the fibroblasts, are responsible for intimal collagenization typically found in atherosclerosis. SMCs, in addition to having an established role in vasoconstriction and dilation, are capable of synthesizing various types of collagens, elastins and proteoglycans. Cells migrate to sites of injury, proliferate and further respond to that injury by secreting a large variety of substances. SMCs also have receptors for low-density lipoproteins as well as enzymes that regulate intracellular cholesterol metabolism. Although not normally phagocytic, SMCs can be stimulated to perform pinocytosis and phagocytosis and to develop a variety of hydrolytic enzymes. These processes may be important in lipid accumulation in vessel walls during atherosclerosis.
Vessels may be damaged due to direct injury or disease and are, of course, also affected by lesions of surrounding tissues which may also be caused by injury or disease. All vascular diseases are similar in that they damage the vessel walls, leading to dilation or rupture, narrow the lumina of the vessel producing ischemia, or damage the endothelial lining provoking intravascular thrombosis. Vascular disorders include varicose veins, which is more debilitating than life-threatening, phlebothrombosis which can lead to death through embolism, congenital anomalies such as arteriovenous fistula and aneurysm, and a wide variety of tumors and other neoplasms.
One prevalent and clinically significant vessel disease is arteriosclerosis. In time, this disorder, to some degree, affects nearly every individual. Arteriosclerosis quite literally means, hardening of the arteries. This disorder more accurately refers to a group of disorders that have a common thickening and loss of elasticity of arterial walls. The three distinct morphologies, characterized by formation of fibro-fatty intimal plaques (atheromas) and all referred to as atherosclerosis, include Monckeberg's medical calcific sclerosis, characterized by calcification of the media of muscular arteries, and atherosclerosis, characterized by proliferation or hyaline thickening of the walls of the small arteries and arterioles. More than one of these disorders is typically found in a single individual.
SMCs, the cells responsible for the production of connective tissue, the bulk of the vessel wall and to a large extent vessel wall integrity, demonstrate increased migration and proliferation within the intima of diseased and injured arteries. These cells can be characterized morphologically as unstriated and spindle-shaped with centrally located nuclei. Typically, smooth muscle cells are bound in sheets and are found in the internal organs including the uterus and heart, hair follicles and, of course, blood vessels. Proliferation of SMCs within vessels results in excess connective tissue deposition and the development of atherosclerotic fibrous plaques that block veins and arteries producing pathologies such as atherosclerosis, hypertension, ischemic injury, stroke, and myocardial infarction. The formation of plaques within vessels leads to partial blockage or occlusion of blood flow, also referred to as stenosis, to major arteries and tissues. Proliferation of SMCs into and along the vessel walls, to a large extent, is responsible for plaque formation. Pathology is not limited to arteries, but can also occur in any vessel including veins and arterioles.
Injury of the vascular endothelium is considered by most to be the initiating event in the development of stenosis. Mechanical injuries such as angioplasty, vascular surgery, transplantation surgery and other invasive processes that disrupt vascular integrity, lead to the proliferation of smooth muscle cells in vessels and arteries. Stenosis is also induced biologically by stresses, which may be internally or externally derived, that injure the vascular endothelium.
Current treatment regimes for stenosis or occluded vessels includes mechanical interventions, however, these techniques also serve to exacerbate the injury, precipitating a new bout of SMC proliferation. For example, occluded arteries are often treated with balloon angioplasty which involves the mechanical dilation of a vessel with an inflatable catheter. The effectiveness of this surgery is limited because the treatment itself damages the vessel thereby inducing proliferation of SMCs and re-occlusion or restenosis of the vessel. Approximately 30-40% of patients treated by balloon angioplasty experience restenosis within one year of surgery.
A number of agents which affect cell proliferation have been tested as pharmacological treatments for stenosis and restenosis in an attempt to slow or inhibit proliferation of SMCs. These compositions have included heparin, coumarin, aspirin, fish oils, calcium antagonists, steroids, prostacyclin, rapamycin, dipryidamole, ultraviolet irradiation, gamma (.gamma.)-interferon, serotonin inhibitors, methotextrate and mycophenolic acid, either alone or in various combinations. For example, heparin is commonly used following coronary angioplasty to reduce the incidence of acute thrombotic occlusion and reduce the proliferation of SMCs (Guyton et al., Circ. Res. 46:625, 1980). These activities were demonstrated in vitro and confirmed in vivo in experiments on rat arterial SMC proliferation after balloon catheter injury (Gordon et al., Circulation 76:213, 1987). Wai et al. determined that a hybrid protein consisting of the ribosome inhibitor, saponin, fused to basic fibroblast growth factor (FGF), killed proliferating FGF-receptor expressing SMCs, but not quiescent receptor negative cells (Wai et al., Circulation 82:208, 1990). This same hybrid protein also inhibited intimal thickening following vascular injury.
Acetylsalicylic acid pre-treatment has been shown to reduce platelet accumulation in patients who have undergone coronary angioplasty (Cunningham et al., Radiology 151:487, 1984). A placebo controlled study in 376 patients demonstrated that while an aspirin-dipyridamine, anti-platelet regimen before and after percutaneous transluminal coronary angioplasty did not reduce the six-month rate of restenosis after successful coronary angioplasty, it markedly reduced the incidence of transmural myocardial infarction during or soon after percutaneous transluminal coronary angioplasty (Schwartz et al., N. Engl. J. Med. 318:1714, 1988).
Agents that interfere with the action of certain cytokines have also been tested for their effect on stenosis. For example, U.S. Pat. No. 5,268,358 is directed to the use of peptides that block the binding of platelet derived growth factors to their receptors. U.S. Pat. No. 5,304,541 is directed to chimeric transforming growth factor-beta (TGF-.beta.) peptides which block cell proliferation. U.S. Pat. No. 5,308,622 is directed to conjugates comprising fibroblastic growth factor (FGF) and cytotoxic agents. U.S. Pat. No. 5,326,559 is directed to interleukin-2 targeted molecules. Although promising, many of these agents and compositions have known and serious side effects and, consequently, limited effectiveness. Each of these U.S. patents is hereby specifically incorporated by reference.
Successful treatment with pharmaceuticals generally requires delivery of the active agent to the site of the injury or the site responsible for the injury. Systemic delivery can involve enteral or parenteral routes of administration. Oral ingestion is the most common method of drug administration and also the safest. Disadvantages include the inability of certain compositions or agents to be absorbed through the gastrointestinal mucosa and destruction by the low gastric pH and enzymes present in the gastrointestinal tract. Drugs which are administered in this manner also often metabolize before they gain access to the blood stream and have an opportunity to produce any sort of beneficial effect.
Parenteral administration includes, for example, topical application to dermal tissues, pulmonary absorption (U.S. Pat. No. 5,241,049), and direct injection into the blood stream or some other site of the body, thereby avoiding the harsh environment of the gastrointestinal tract. Although topical applications are generally safe, direct injections carry a number of significant risks. Injections necessarily create a hole in the outer integument of the body providing an entrance for bacteria, virus particles and toxic substances. Consequently, aseptic conditions must be maintained for all types of injections including intravenous, subcutaneous and intramuscular.
Local delivery of a pharmaceutical agent is becoming increasingly popular and possible. Proteins have been delivered to site specific locations in the body by implanting biodegradable polymer matrices containing the pharmaceutical as described in U.S. Pat. Nos. 5,328,695 and 5,271,961. As the polymer degrades, the resulting degraded components, which are safe and non-toxic, are easily absorbed or eliminated by the body. More importantly, the released agent has an opportunity to perform its intended function before being inactivated. This method is especially useful for delivery of agents with short half-lives which would be otherwise ineffective even if they were able to pass through the gastric mucosa. Other somewhat similar methods include magnetic release of agents (U.S. Pat. No. 5,125,888), fusion to conjugates capable of rapid absorption into desired cells (U.S. Pat. Nos. 5,324,655 and 5,254,342), and charge modification of the active component (U.S. Pat. No. 5,322,678). Each of these U.S. patents is hereby specifically incorporated by reference.
Local delivery has also been achieved with the use of catheters (U.S. Pat. No. 4,636,195), stents (U.S. Pat. No. 5,304,121), coatings on balloon catheters (U.S. Pat. No. 5,102,402), direct injection of the agent formulated with a biodegradable polymer (U.S. Pat. No. 5,171,217), and hydrogel polymer/agent coatings on catheters. These anti-thrombogenic and anti-proliferative agents have demonstrated limited successes.
Cell proliferation in stenosis results, at least in part, from the actions of a variety of growth factors and cytokines. These include platelet derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and a variety of interleukins (IL-1 and IL-2). One of the nuclear factors found associated with cells is the transcription factor NF-.kappa.B, a member of the rel-family of factors. NF-.kappa.B was first identified as an activity that specifically retarded the mobility of DNA fragments containing the decameric sequence 5'-GGGACTTTCC-3' (SEQ ID NO 1) (R. Sen et al., Cell 46:705-16, 1986). This sequence was identified as a B cell-specific binding element in the kappa (.kappa.) light chain immunoglobulin gene enhancer (R. Sen et al., Cell 47:921-28, 1986). NF-.kappa.B binding to this element was induced during pre-B to B cell differentiation in association with the activation of .kappa. light chain gene transcription. HIV-1 LTR contains two NF-.kappa.B elements that are important for transcription of this promoter (G. Nabel et al., Nature 326:711-13, 1987).
The presence of NF-.kappa.B protein was found to be fairly ubiquitous in many different cell types. In most cells this protein is sequestered in the cytoplasm with an inhibitor protein referred to as I.kappa.B (P. A. Baeuerle et al., Science 242:540-46, 1988). Activation and nuclear localization can be induced in these cells by several agents. Activating agents include phorbol ester, IL-1, TNF-.alpha., ultraviolet light, and serum, as well as infection by a number of viruses, including the human T-cell leukemia virus type I and Epstein-Barr viruses. Activation involves post-translational events, including inactivation and rapid degradation of I.kappa.B (K. Brown et al., Proc. Natl. Acad. Sci., USA 90:2532-36, 1993).
In addition to being required for activation of the .kappa. light chain gene, NF-.kappa.B has been implicated in control of transcription of a number of cellular genes involved in immune and inflammatory responses, growth and adhesion (M. Grilli et al., Int. Rev. Cytol. 143:1-62, 1993). Some of these genes encode interleukins or their receptors, such as IL-2, IL-2R.alpha.IL-6, IL-8, class I MHC, GM-CSF and TNF-.beta., the SAA acute phase response gene, .beta.-interferon, gro, and V-CAM-I. The c-myc oncogene contains two NF-.kappa.B binding elements called the upstream regulatory element (URE) and the internal regulatory element (IRE) (M. P. Duyao et al., Proc. Natl. Acad. Sci. USA 87:4727-31, 1990; D. J. Kessler et al., Oncogene 7:2447-53, 1992). Both sites have been shown to be required for normal activation of c-myc expression by NF-.kappa.B. NF-.kappa.B is also involved in the control of the expression of receptors, adhesion molecules and components of the cytoskeleton.
Biochemical characterization of classical NF-.kappa.B demonstrated that it is a heterodimer composed of a 50 kD (p50) subunit and a 65 kD (p65) subunit (S. Ghosh et al., Cell 62:1019-29, 1990; M. Kieran et al., Cell 62:1007-18, 1990; S. Ruben et al., Science 251:490-93, 1991). Cloning and sequencing of p50 and p65 led to the discovery that these factors are members of a larger family of proteins that includes the rel oncoproteins and the Drosophila dorsal gene products (M. Grilli et al., Internat'l Rev. Cytol. 143:1-62, 1993). The rel-related family includes p50, p65, p52 (V. Bours et al., Mol. Cell. Biol. 12:685-95, 1992). c-rel, v-rel and rel-B (R. P. Ryseck et al., Mol. Cell. Biol. 12:674-84, 1992). Rel-related family members have been shown to regulate gene expression for a number of viruses including cytomegalovirus (CMV), and been recently implicated in the formation of atherosclerotic plaques (P. A. Baeuerle, Biochem. Biophys. Acta 1072:63-80, 1991; R. Ross, Nature 362:801-08, 1993), and to be involved with proliferation and restenosis (E. Speir et al., Sci. 265:391-94, 1994).
Rel-related factors generally bind to DNA as dimers (M. B. Urban et al., EMBO J. 10:1817-25, 1992). The p65 subunit, which binds to the 3' side of the binding element, contains a potent transcription activation domain (M. L. Schmitz et al., EMBO J. 10:3805-17, 1991). Transfections of vector expressing both p50 and p65, or p65 alone activate transcription of the c-myc promoter (F. La Rosa et al., Mol. Cell. Biol. 14:1039-44, 1994). In contrast, the p50 subunit alone is unable to activate transcription in vivo or activates only weakly in most cell types. c-rel, another Rel-related protein which appears to function in an element specific fashion, activates transcription moderately (T.-H. Tan et al., Mol. Cell. Biol. 12:4067-75, 1992; P. McDonell et al., Oncogene 7:163-170, 1992), whereas rel B significantly activates transcription (P. Dobrzanski et al., Mol. Cell. Biol. 13:1572-82, 1993).
Constitutive activity of this family of factors was previously thought to be restricted to hematopoietic lineage cells such as mature B cells, thymocytes and macrophages (M. J. Lenardo et al., Cell 58:227-31, 1989; M. Korner et al., Biochem. Biophys. Res. Commun. 181:80-86, 1991). However, activity of rel-related factors has been reported recently in cells of neural origin (C. Kaltschmidt et al., Mol. Cell. Biol. 14:3981-3992, 1994), and a novel inducible factor was identified in the liver (M. Tewari et al., Mol. Cell. Biol. 12:2898-2908, 1992).